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US20090117127A1 - Novel Compounds and Methods for Their Production - Google Patents

Novel Compounds and Methods for Their Production Download PDF

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Publication number
US20090117127A1
US20090117127A1 US12/294,099 US29409907A US2009117127A1 US 20090117127 A1 US20090117127 A1 US 20090117127A1 US 29409907 A US29409907 A US 29409907A US 2009117127 A1 US2009117127 A1 US 2009117127A1
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desmethylmacbecin
strain
analogue
mbcmt1
macbecin
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Inventor
Sabine Gaisser
Christine Martin
Ming Zhang
Barrie Wilkinson
Lesley Sheehan
Nigel Coates
Mohammed Nur-E-Alam
William Vousden
Nikolaos Gaitatzis
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Biotica Technology Ltd
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Biotica Technology Ltd
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Assigned to BIOTICA TECHNOLOGY LIMITED reassignment BIOTICA TECHNOLOGY LIMITED ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SHEEHAN, LESLEY, COATES, NIGEL, MARTIN, CHRISTINE JANET, NUR-E-ALAM, MOHAMMED, WILKINSON, BARRIE, ZHANG, MING-QIANG, GAISSER, SABINE, VOUSDEN, WILLIAM, GAITATZIS, NIKOLAOS
Publication of US20090117127A1 publication Critical patent/US20090117127A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D225/00—Heterocyclic compounds containing rings of more than seven members having one nitrogen atom as the only ring hetero atom
    • C07D225/04—Heterocyclic compounds containing rings of more than seven members having one nitrogen atom as the only ring hetero atom condensed with carbocyclic rings or ring systems
    • C07D225/06—Heterocyclic compounds containing rings of more than seven members having one nitrogen atom as the only ring hetero atom condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Hsp90 The 90 kDa heat shock protein
  • So far nearly 50 of these so-called client proteins have been identified and include steroid receptors, non-receptor tyrosine kinases e.g. src family, cyclin-dependent kinases e.g.
  • Hsp90 plays a key role in stress response and protection of the cell against the effects of mutation (Bagatell and Whitesell, 2004; Chiosis et al., 2004).
  • Hsp90 The function of Hsp90 is complicated and it involves the formation of dynamic multi-enzyme complexes (Bohen, 1998; Liu et al., 1999; Young et al., 2001; Takahashi et al., 2003; Sreedhar et al., 2004; Wegele et al., 2004).
  • Hsp90 is a target for inhibitors (Fang et al., 1998; Liu et al., 1999; Blagosklonny, 2002; Neckers, 2003; Takahashi et al., 2003; Beliakoff and Whitesell, 2004; Wegele et al., 2004) resulting in degradation of client proteins, cell cycle dysregulation and apoptosis.
  • Hsp90 has been identified as an important extracellular mediator for tumour invasion (Eustace et al., 2004). Hsp90 was identified as a new major therapeutic target for cancer therapy which is mirrored in the intense and detailed research about Hsp90 function (Blagosklonny et al., 1996; Neckers, 2002; Workman and Kaye, 2002; Beliakoff and Whitesell, 2004; Harris et al., 2004; Jez et al., 2003; Lee et al., 2004) and the development of high-throughput screening assays (Carreras et al., 2003; Rowlands et al., 2004).
  • Hsp90 inhibitors include compound classes such as ansamycins, macrolides, purines, pyrazoles, coumarin antibiotics and others (for review see Bagatell and Whitesell, 2004; Chiosis et al., 2004 and references therein).
  • the benzenoid ansamycins are a broad class of chemical structures characterised by an aliphatic ring of varying length joined either side of an aromatic ring structure.
  • Naturally occurring ansamycins include: macbecin and 18,21-dihydromacbecin (also known as macbecin I and macbecin II respectively) (1 & 2; Tanida et al., 1980), geldanamycin (3; DeBoer et al., 1970; DeBoer and Dietz, 1976; WO 03/106653 and references therein), and the herbimycin family (4; 5, 6, Omura et al., 1979, Iwai et al., 1980 and Shibata et al, 1986a, WO 03/106653 and references therein).
  • geldanamycin has nanomolar potency and apparent specificity for aberrant protein kinase dependent tumour cells (Chiosis et al., 2003; Workman, 2003).
  • Hsp90 inhibitors enhances the induction of tumour cell death by radiation and increased cell killing abilities (e.g. breast cancer, chronic myeloid leukaemia and non-small cell lung cancer) by combination of Hsp90 inhibitors with cytotoxic agents has also been demonstrated (Neckers, 2002; Beliakoff and Whitesell, 2004).
  • cytotoxic agents e.g. IL-12, IL-12, IL-12, IL-12, IL-12
  • Hsp90 inhibitors also function as immunosuppressants and are involved in the complement-induced lysis of several types of tumour cells after Hsp90 inhibition (Sreedhar et al., 2004). Treatment with Hsp90 inhibitors can also result in induced superoxide production (Sreedhar et al., 2004a) associated with immune cell-mediated lysis (Sreedhar et al., 2004).
  • Hsp90 inhibitors as potential anti-malaria drugs has also been discussed (Kumar et al., 2003).
  • geldanamycin interferes with the formation of complex glycosylated mammalian prion protein PrP c (Winklhofer et al., 2003).
  • ansamycins are of interest as potential anticancer and anti-B-cell malignancy compounds, however the currently available ansamycins exhibit poor pharmacological or pharmaceutical properties, for example they show poor water solubility, poor metabolic stability, poor bioavailability or poor formulation ability (Goetz et al., 2003; Workman 2003; Chiosis 2004). Both herbimycin A and geldanamycin were identified as poor candidates for clinical trials due to their strong hepatotoxicity (review Workman, 2003) and geldanamycin was withdrawn from Phase I clinical trials due to hepatotoxicity (Supko et al., 1995; WO 03/106653).
  • Geldanamycin was isolated from culture filtrates of Streptomyces hygroscopicus and shows strong activity in vitro against protozoa and weak activity against bacteria and fungi. In 1994 the association of geldanamycin with Hsp90 was shown (Whitesell et al., 1994). The biosynthetic gene cluster for geldanamycin was cloned and sequenced (Allen and Ritchie, 1994; Rascher et al., 2003; WO 03/106653). The DNA sequence is available under the NCBI accession number AY179507. The isolation of genetically engineered geldanamycin producer strains derived from S. hygroscopicus subsp.
  • duamyceticus JCM4427 and the isolation of 4,5-dihydro-7-O-descarbamoyl-7-hydroxygeldanamycin and 4,5-dihydro-7-O-descarbamoyl-7-hydroxy-17-O-demethylgeldanamycin were described recently (Hong et al., 2004).
  • geldanamycin By feeding geldanamycin to the herbimycin producing strain Streptomyces hygroscopicus AM-3672 the compounds 15-hydroxygeldanamycin, the tricyclic geldanamycin analogue KOSN-1633 and methyl-geldanamycinate were isolated (Hu et al., 2004).
  • S. hygroscopicus K279-78 is S. hygroscopicus NRRL 3602 containing cosmid pKOS279-78 which has a 44 kbp insert which contains various genes from the herbimycin producing strain Streptomyces hygroscopicus AM-3672 (Hu et al., 2004).
  • ansamycin antibiotic herbimycin A was isolated from the fermentation broth of Streptomyces hygroscopicus strain No. AM-3672 and named according to its potent herbicidal activity.
  • the antitumour activity was established by using cells of a rat kidney line infected with a temperature sensitive mutant of Rous sarcoma virus (RSV) for screening for drugs that reverted the transformed morphology of the these cells (for review see Uehara, 2003).
  • RSV Rous sarcoma virus
  • Herbimycin A was postulated as acting primarily through the binding to Hsp90 chaperone proteins but the direct binding to the conserved cysteine residues and subsequent inactivation of kinases was also discussed (Uehara, 2003).
  • the ansamycin compounds macbecin (1) and 18,21-dihydromacbecin (2) (C-14919E-1 and C-14919E-1), identified by their antifungal and antiprotozoal activity, were isolated from the culture supernatants of Nocardia sp No. C-14919 ( Actinosynnema pretiosum subsp pretiosum ATCC 31280) (Tanida et al., 1980; Muroi et al., 1980; Muroi et al., 1981; U.S. Pat. No. 4,315,989 and U.S. Pat. No. 4,187,292).
  • 18,21-Dihydromacbecin is characterized by containing the dihydroquinone form of the nucleus.
  • the compounds TAN-420A to E were identified from producer strains belonging to the genus Streptomyces (7-11, EP 0 110 710).
  • a further Hsp90 inhibitor, distinct from the chemically unrelated benzoquinone ansamycins is Radicicol (monorden) which was originally discovered for its antifungal activity from the fungus Monosporium bonorden (for review see Uehara, 2003) and the structure was found to be identical to the 14-membered macrolide isolated from Nectria radicicola . In addition to its antifungal, antibacterial, anti-protozoan and cytotoxic activity it was subsequently identified as an inhibitor of Hsp90 chaperone proteins (for review see Uehara, 2003; Schulte et al., 1999). The anti-angiogenic activity of radicicol (Hur et al., 2002) and semi-synthetic derivates thereof (Kurebayashi et al., 2001) has also been described.
  • geldanamycin was derivatised on the 17-position to create 17-geldanamycin amides, carbamates, ureas and 17-arylgeldanamycin (Le Brazidec et al., 2003).
  • a library of over sixty 17-alkylamino-17-demethoxygeldanamycin analogues has been reported and tested for their affinity for Hsp90 and water solubility (Tian et al., 2004).
  • a further approach to reduce the toxicity of geldanamycin is the selective targeting and delivering of an active geldanamycin compound into malignant cells by conjugation to a tumour-targeting monoclonal antibody (Mandler et al., 2000).
  • 17-AAG requires the use of a solubilising carrier (e.g. Cremophore®, DMSO-egg lecithin), which itself may result in side-effects in some patients (Hu et al., 2004).
  • a solubilising carrier e.g. Cremophore®, DMSO-egg lecithin
  • ansamycin class of Hsp90 inhibitors bear the common structural moiety: the benzoquinone which is a Michael acceptor that can readily form covalent bonds with nucleophiles such as proteins, glutathione, etc.
  • the benzoquinone moiety also undergoes redox equilibrium with dihydroquinone, during which oxygen radicals are formed, which give rise to further unspecific toxicity (Dikalov et al., 2002).
  • treatment with geldanamycin can result in induced superoxide production (Sreedhar et al., 2004a).
  • novel ansamycin derivatives which may have utility in the treatment of cancer and/or B-cell malignancies, preferably such ansamycins have improved water solubility, an improved pharmacological profile and/or reduced side-effect profile for administration.
  • the present invention discloses novel ansamycin analogues generated by genetic engineering of the parent producer strain.
  • novel 11-O-desmethylmacbecin analogues which generally have improved pharmaceutical properties compared with the presently available ansamycins; in particular they are expected show improvements in respect of one or more of the following properties: activity against different cancer sub-types, toxicity, water solubility, metabolic stability, bioavailability and formulation ability.
  • the 11-O-desmethylmacbecin analogues show improved water solubility and/or bioavailability.
  • the inventors of the present invention have made significant effort to clone and elucidate the gene cluster that is responsible for the biosynthesis of macbecin.
  • the gene that is responsible for the addition of a methyl group to the oxygen attached at the C11 position has been specifically targeted, e.g. by integration into mbcMT1, targeted deletion of a region of the macbecin cluster including all or part of the mbcMT1 gene optionally followed by insertion of gene(s) or other methods of rendering MbcMT1 non-functional e.g.
  • the present invention provides 11-O-desmethylmacbecin analogues, methods for the preparation of these compounds, and methods for the use of these compounds in medicine or as intermediates in the production of further compounds.
  • the present invention provides analogues of macbecin which are lacking the methyl group usually attached to an oxygen at the C11 position
  • the macbecin analogues may either have a benzoquinone (i.e. they are macbecin I analogues) or have a dihydroquinone moiety (i.e., they are 18,21-dihydromacbecin or macbecin II analogues).
  • the present invention provides 11-O-desmethylmacbecin analogues according to the formula (IA) or (IB) below, or a pharmaceutically acceptable salt thereof:
  • R 1 represents H, OH, OMe
  • R 2 represents H or CONH 2 ;
  • R 3 and R 4 either both represent H or together they represent a bond (i.e. C4 to C5 is a double bond)
  • 11-O-Desmethylmacbecin analogues are also referred to herein as “compounds of the invention”, such terms are used interchangeably herein.
  • Compounds of formula (IA) and (IB) are referred to collectively in the foregoing as compounds of formula (I).
  • the invention embraces all stereoisomers of the compounds defined by structure (I) as shown above.
  • the present invention provides 11-O-desmethylmacbecin analogues such as compounds of formula (I) or a pharmaceutically acceptable salt thereof, for use as a pharmaceutical.
  • analogue means one analogue or more than one analogue.
  • analogue(s) refers to chemical compounds that are structurally similar to another but which differ slightly in composition (as in the replacement of one atom by another or in the presence or absence of a particular functional group).
  • homologue(s) refers a homologue of a gene or of a protein encoded by a gene disclosed herein from either an alternative macbecin biosynthetic cluster from a different macbecin producing strain or a homologue from an alternative ansamycin biosynthetic gene cluster e.g. from geldanamycin, herbimycin or reblastatin.
  • Such homologue(s) encode a protein that performs the same function of can itself perform the same function as said gene or protein in the synthesis of macbecin or a related ansamycin polyketide.
  • such homologue(s) have at least 40% sequence identity, preferably at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to the sequence of the particular gene disclosed herein (Table 3, SEQ ID NO: 11 which is a sequence of all the genes in the cluster, from which the sequences of particular genes may be deduced). Percentage identity may be calculated using any program known to a person of skill in the art such as BLASTn or BLASTp, available on the NCBI website.
  • cancer refers to a benign or malignant new growth of cells in skin or in body organs, for example but without limitation, breast, prostate, lung, kidney, pancreas, brain, stomach or bowel.
  • a cancer tends to infiltrate into adjacent tissue and spread (metastasise) to distant organs, for example to bone, liver, lung or the brain.
  • cancer includes both metastatic tumour cell types, such as but not limited to, melanoma, lymphoma, leukaemia, fibrosarcoma, rhabdomyosarcoma, and mastocytoma and types of tissue carcinoma, such as but not limited to, colorectal cancer, prostate cancer, small cell lung cancer and non-small cell lung cancer, breast cancer, pancreatic cancer, bladder cancer, renal cancer, gastric cancer, gliobastoma, primary liver cancer and ovarian cancer.
  • metastatic tumour cell types such as but not limited to, melanoma, lymphoma, leukaemia, fibrosarcoma, rhabdomyosarcoma, and mastocytoma
  • types of tissue carcinoma such as but not limited to, colorectal cancer, prostate cancer, small cell lung cancer and non-small cell lung cancer, breast cancer, pancreatic cancer, bladder cancer, renal cancer, gastric cancer, gliobastoma, primary liver cancer and ovarian cancer.
  • B-cell malignancies includes a group of disorders that include chronic lymphocytic leukaemia (CLL), multiple myeloma, and non-Hodgkin's lymphoma (NHL). They are neoplastic diseases of the blood and blood forming organs. They cause bone marrow and immune system dysfunction, which renders the host highly susceptible to infection and bleeding.
  • CLL chronic lymphocytic leukaemia
  • NHL non-Hodgkin's lymphoma
  • bioavailability refers to the degree to which or rate at which a drug or other substance is absorbed or becomes available at the site of biological activity after administration. This property is dependent upon a number of factors including the solubility of the compound, rate of absorption in the gut, the extent of protein binding and metabolism etc.
  • tests for bioavailability that would be familiar to a person of skill in the art are for example described in Egorin et al. (2002).
  • water solubility refers to solubility in aqueous media, e.g. phosphate buffered saline (PBS) at pH 7.3.
  • PBS phosphate buffered saline
  • An exemplary water solubility assay is given in the Examples below.
  • post-PKS genes(s) refers to the genes required for post-polyketide synthase modifications of the polyketide, for example but without limitation monooxygenases, O-methyltransferases and carbamoyltransferases.
  • these modifying genes include mbcM, mbcN, mbcP, mbcMT1, mbcMT2 and mbcP450.
  • the pharmaceutically acceptable salts of compounds of the invention include conventional salts formed from pharmaceutically acceptable inorganic or organic acids or bases as well as quaternary ammonium acid addition salts. More specific examples of suitable acid salts include hydrochloric, hydrobromic, sulfuric, phosphoric, nitric, perchloric, fumaric, acetic, propionic, succinic, glycolic, formic, lactic, maleic, tartaric, citric, palmoic, malonic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, fumaric, toluenesulfonic, methanesulfonic, naphthalene-2-sulfonic, benzenesulfonic hydroxynaphthoic, hydroiodic, malic, steroic, tannic and the like.
  • acids such as oxalic, while not in themselves pharmaceutically acceptable, may be useful in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable salts.
  • suitable basic salts include sodium, lithium, potassium, magnesium, aluminium, calcium, zinc, N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine and procaine salts.
  • References hereinafter to a compound according to the invention include both compounds of formula (I) and their pharmaceutically acceptable salts.
  • FIG. 1 Representation of the biosynthesis of macbecin showing the first putative enzyme free intermediate, pre-macbecin and the post-PKS processing to macbecin.
  • the list of PKS processing steps in the figure in not intended to represent the order of events.
  • the following abbreviations are used for particular genes in the cluster: AL0—AHBA loading domain; ACP—Acyl Carrier Protein; KS— ⁇ -ketosynthase; AT—acyl transferase; DH—dehydratase; ER—enoyl reductase; KR— ⁇ -ketoreductase.
  • FIG. 2 Depiction of the sites of post-PKS processing of pre-macbecin to give macbecin.
  • FIG. 3 Diagrammatic representation of generation of the engineered strain BIOT-3820 in which plasmid pGP24 was integrated into the chromosome by homologous recombination resulting in mbcMT1 gene disruption.
  • FIG. 4 Sequence of the amplified PCR product PCRgp24 (SEQ ID NO: 14).
  • FIG. 5 Sequence of the 518 bp internal DNA fragment of mbcP450 (SEQ ID NO: 17)
  • FIG. 6 Diagrammatic representation of generation of the engineered strain BIOT-3825 in which plasmid pNGmbcP450 was integrated into the chromosome by homologous recombination resulting in mbcP450 gene disruption.
  • FIG. 7 Sequence of the amplified PCR product PCR1 (SEQ ID NO: 20)
  • FIG. 8 Sequence of the amplified PCR product PCR2 (SEQ ID NO: 24)
  • FIG. 9 Diagrammatic representation of the generation of an Actinosynnema pretiosum strain carrying a deletion of the methyltransferase genes mbcMT1 and mbcMT2.
  • FIG. 10 Sequence of an 801 bp region of DNA, PCR mbcMT2 (SEQ ID NO: 28)
  • FIG. 11 Diagrammatic representation of the generation of an Actinosynnema pretiosum strain in which the mbcP, mbcP450, mbcMT1 and mbcMT2 genes have been deleted in frame.
  • FIG. 12 Sequence of the amplified PCR product 1+2a (SEQ ID NO: 31)
  • FIG. 13 Sequence of the amplified PCR product 3b+4 (SEQ ID NO: 34)
  • FIG. 14 Sequence of a 526 bp internal DNA fragment of mbcP (SEQ ID NO: 37)
  • FIG. 15 Diagrammatic representation of the generation of BIOT-3863; an Actinosynnema pretiosum strain in which the mbcP has been interrupted by insertion of a plasmid.
  • FIG. 16 Structures of the compounds (14-17) produced in the examples.
  • the present invention provides 11-O-desmethylmacbecin analogues, as set out above, methods for the preparation of these compounds, methods for the use of these compounds in medicine and the use of these compounds as intermediates or templates for further semi-synthetic derivatisation or derivatisation by biotransformation methods.
  • the 11-O-desmethylmacbecin analogues have a structure according to Formula IA.
  • the 11-O-desmethylmacbecin analogues have a structure according to Formula IB.
  • R 2 represents CONH 2
  • R 3 and R 4 together represent a bond
  • R 1 represents OCH 3
  • R 2 represents CONH 2
  • R 3 and R 4 together represent a bond
  • R 1 represents OH
  • R 2 represents CONH 2
  • R 3 and R 4 together represent a bond
  • R 1 represents H
  • R 2 represents CONH 2
  • R 3 and R 4 together represent a bond
  • R 1 represents H
  • R 2 represents CONH 2
  • R 3 and R 4 each represent H.
  • the preferred stereochemistry of the non-hydrogen sidechains to the ansa ring is as shown in FIGS. 1 , 2 and 16 below (that is to say the preferred stereochemistry follows that of macbecin).
  • the compounds of the invention may be isolated from the fermentation broth in their benzoquinone form or in their dihydroquinone form. It is well-known in the art that benzoquinones can be chemically converted to dihydroquinones (reduction) and vice versa (oxidation), therefore these forms may be readily interconverted using methods well-known to a person of skill in the art. For example, but without limitation, if the benzoquinone form is isolated then it may be converted to the corresponding dihydroquinone. As an example (but not by way of limitation) this may be achieved in organic media with a source of hydride, such as but not limited to, LiAlH 4 or SnCl 2 —HCl.
  • a source of hydride such as but not limited to, LiAlH 4 or SnCl 2 —HCl.
  • this transformation may be mediated by dissolving the benzoquinone form of the compound of the invention in organic media and then washing with an aqueous solution of a reducing agent, such as, but not limited to, sodium hydrosulfite (Na 2 S 2 O 4 or sodium thionite).
  • a reducing agent such as, but not limited to, sodium hydrosulfite (Na 2 S 2 O 4 or sodium thionite).
  • this transformation is carried out by dissolving the compound of the invention in ethyl acetate and mixing this solution vigorously with an aqueous solution of sodium hydrosulfite (Muroi et al., 1980).
  • the resultant organic solution can then be washed with water, dried and the solvent removed under reduced pressure to yield an almost quantitative amount of the 18,21-dihydro form of the compound of the invention.
  • the dihydroquinone form of the compound of the invention is dissolved in an organic solvent such as ethyl acetate and then this solution is vigorously mixed with an aqueous solution of iron (III) chloride (FeCl 3 ).
  • the organic solution can then be washed with water, dried and the organic solvent removed under reduced pressure to yield an almost quantitative amount of the benzoquinone form of the macbecin compound.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an 11-O-desmethylmacbecin analogue, or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier.
  • the present invention also provides for the use of an 11-O-desmethylmacbecin analogue as a substrate for further modification either by biotransformation or by synthetic chemistry.
  • the present invention provides for the use of an 11-O-desmethylmacbecin analogue in the manufacture of a medicament.
  • the present invention provides for the use of an 11-O-desmethylmacbecin analogue in the manufacture of a medicament for the treatment of cancer and/or B-cell malignancies.
  • the present invention provides for the use of an 11-O-desmethylmacbecin analogue in the manufacture of a medicament for the treatment of malaria, fungal infection, diseases of the central nervous system, diseases dependent on angiogenesis, autoimmune diseases and/or as a prophylactic pre-treatment for cancer.
  • the present invention provides for the use of an 11-O-desmethylmacbecin analogue in medicine.
  • the present invention provides for the use of an 11-O-desmethylmacbecin analogue in the treatment of cancer and/or B-cell malignancies.
  • the present invention provides for the use of an 11-O-desmethylmacbecin analogue in the manufacture of a medicament for the treatment of malaria, fungal infection, diseases of the central nervous system and neurodegenerative diseases, diseases dependent on angiogenesis, autoimmune diseases and/or as a prophylactic pre-treatment for cancer.
  • the present invention provides a method of treatment of cancer and/or B-cell malignancies, said method comprising administering to a patient in need thereof a therapeutically effective amount of an 11-O-desmethylmacbecin analogue.
  • the present invention provides a method of treatment of malaria, fungal infection, diseases of the central nervous system and neurodegenerative diseases, diseases dependent on angiogenesis, autoimmune diseases and/or a prophylactic pre-treatment for cancer, said method comprising administering to a patient in need thereof a therapeutically effective amount of an 11-O-desmethylmacbecin analogue.
  • compounds of the invention may be expected to be useful in the treatment of cancer and/or B-cell malignancies.
  • Compounds of the invention may also be effective in the treatment of other indications for example, but not limited to malaria, fungal infection, diseases of the central nervous system and neurodegenerative diseases, diseases dependent on angiogenesis, autoimmune diseases such as rheumatoid arthritis or as a prophylactic pre-treatment for cancer.
  • ALS amyotrophic lateral sclerosis
  • Diseases dependent on angiogenesis include, but are not limited to, age-related macular degeneration, diabetic retinopathy and various other ophthalmic disorders, atherosclerosis and rheumatoid arthritis.
  • Autoimmune diseases include, but are not limited to, rheumatoid arthritis, multiple sclerosis, type I diabetes, systemic lupus erythematosus and psoriasis.
  • “Patient” embraces human and other animal (especially mammalian) subjects, preferably human subjects. Accordingly the methods and uses of the 11-O-desmethylmacbecin analogues of the invention are of use in human and veterinary medicine, preferably human medicine.
  • the aforementioned compounds of the invention or a formulation thereof may be administered by any conventional method for example but without limitation they may be administered parenterally (including intravenous administration), orally, topically (including buccal, sublingual or transdermal), via a medical device (e.g. a stent), by inhalation, or via injection (subcutaneous or intramuscular).
  • the treatment may consist of a single dose or a plurality of doses over a period of time.
  • a compound of the invention Whilst it is possible for a compound of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers.
  • a pharmaceutical composition comprising a compound of the invention together with one or more pharmaceutically acceptable diluents or carriers.
  • the diluents(s) or carrier(s) must be “acceptable” in the sense of being compatible with the compound of the invention and not deleterious to the recipients thereof. Examples of suitable carriers are described in more detail below.
  • the compounds of the invention may be administered alone or in combination with other therapeutic agents. Co-administration of two (or more) agents may allow for significantly lower doses of each to be used, thereby reducing the side effects seen. It might also allow resensitisation of a disease, such as cancer, to the effects of a prior therapy to which the disease has become resistant.
  • a pharmaceutical composition comprising a compound of the invention and a further therapeutic agent together with one or more pharmaceutically acceptable diluents or carriers.
  • the present invention provides for the use of a compound of the invention in combination therapy with a second agent e.g. a second agent for the treatment of cancer or B-cell malignancies such as a cytotoxic or cytostatic agent.
  • a second agent e.g. a second agent for the treatment of cancer or B-cell malignancies such as a cytotoxic or cytostatic agent.
  • a compound of the invention is co-administered with another therapeutic agent e.g. a therapeutic agent such as a cytotoxic or cytostatic agent for the treatment of cancer or B-cell malignancies.
  • a therapeutic agent such as a cytotoxic or cytostatic agent for the treatment of cancer or B-cell malignancies.
  • cytotoxic agents such as alkylating agents and mitotic inhibitors (including topoisomerase II inhibitors and tubulin inhibitors).
  • Other exemplary further agents include DNA binders, antimetabolites and cytostatic agents such as protein kinase inhibitors and tyrosine kinase receptor blockers.
  • Suitable agents include, but are not limited to, methotrexate, leukovorin, prenisone, bleomycin, cyclophosphamide, 5-fluorouracil, paclitaxel, docetaxel, vincristine, vinblastine, vinorelbine, doxorubicin (adriamycin), tamoxifen, toremifene, megestrol acetate, anastrozole, goserelin, anti-HER2 monoclonal antibody (e.g. trastuzumab, trade name HerceptinTM), capecitabine, raloxifene hydrochloride, EGFR inhibitors (e.g.
  • gefitinib trade name Iressa®, erlotinib, trade name TarcevaTM, cetuximab, trade name ErbituxTM
  • VEGF inhibitors e.g. bevacizumab, trade name AvastinTM
  • proteasome inhibitors e.g. bortezomib, trade name VelcadeTM
  • imatinib trade name Glivec®.
  • chemotherapeutics such as cisplatin, cytarabine, cyclohexylchloroethylnitrosurea, gemcitabine, Ifosfamid, leucovorin, mitomycin, mitoxantone, oxaliplatin, taxanes including taxol and vindesine; hormonal therapies; monoclonal antibody therapies such as cetuximab (anti-EGFR); protein kinase inhibitors such as dasatinib, lapatinib; histone deacetylase (HDAC) inhibitors such as vorinostat; angiogenesis inhibitors such as sunitinib, sorafenib, lenalidomide; and mTOR inhibitors such as temsirolimus. Additionally, a compound of the invention may be administered in combination with other therapies including, but not limited to, radiotherapy or surgery.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient (compound of the invention) with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • the compounds of the invention will normally be administered orally or by any parenteral route, in the form of a pharmaceutical formulation comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form.
  • a pharmaceutical formulation comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form.
  • the compositions may be administered at varying doses.
  • the compounds of the invention can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications.
  • Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatine and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
  • excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine
  • disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates
  • Solid compositions of a similar type may also be employed as fillers in gelatine capsules.
  • Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols.
  • the compounds of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerine, and combinations thereof.
  • a tablet may be made by compression or moulding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g. povidone, gelatine, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide desired release profile.
  • Formulations in accordance with the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be presented as a bolus, electuary or paste.
  • Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatine and glycerine, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.
  • formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.
  • compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, impregnated dressings, sprays, aerosols or oils, transdermal devices, dusting powders, and the like.
  • These compositions may be prepared via conventional methods containing the active agent.
  • they may also comprise compatible conventional carriers and additives, such as preservatives, solvents to assist drug penetration, emollient in creams or ointments and ethanol or oleyl alcohol for lotions.
  • Such carriers may be present as from about 1% up to about 98% of the composition. More usually they will form up to about 80% of the composition.
  • a cream or ointment is prepared by mixing sufficient quantities of hydrophilic material and water, containing from about 5-10% by weight of the compound, in sufficient quantities to produce a cream or ointment having the desired consistency.
  • compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.
  • the active agent may be delivered from the patch by iontophoresis.
  • compositions are preferably applied as a topical ointment or cream.
  • the active agent may be employed with either a paraffinic or a water-miscible ointment base.
  • the active agent may be formulated in a cream with an oil-in-water cream base or a water-in-oil base.
  • fluid unit dosage forms are prepared utilizing the active ingredient and a sterile vehicle, for example but without limitation water, alcohols, polyols, glycerine and vegetable oils, water being preferred.
  • a sterile vehicle for example but without limitation water, alcohols, polyols, glycerine and vegetable oils, water being preferred.
  • the active ingredient depending on the vehicle and concentration used, can be either suspended or dissolved in the vehicle.
  • the active ingredient can be dissolved in water for injection and filter sterilised before filling into a suitable vial or ampoule and sealing.
  • agents such as local anaesthetics, preservatives and buffering agents can be dissolved in the vehicle.
  • the composition can be frozen after filling into the vial and the water removed under vacuum.
  • the dry lyophilized powder is then sealed in the vial and an accompanying vial of water for injection may be supplied to reconstitute the liquid prior to use.
  • Parenteral suspensions are prepared in substantially the same manner as solutions, except that the active ingredient is suspended in the vehicle instead of being dissolved and sterilization cannot be accomplished by filtration.
  • the active ingredient can be sterilised by exposure to ethylene oxide before suspending in the sterile vehicle.
  • a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the active ingredient.
  • a pharmaceutical composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Pat. No. 5,399,163; U.S. Pat. No. 5,383,851; U.S. Pat. No. 5,312,335; U.S. Pat. No. 5,064,413; U.S. Pat. No. 4,941,880; U.S. Pat. No. 4,790,824; or U.S. Pat. No. 4,596,556.
  • Examples of well-known implants and modules useful in the present invention include: U.S. Pat. No.
  • the dosage to be administered of a compound of the invention will vary according to the particular compound, the disease involved, the subject, and the nature and severity of the disease and the physical condition of the subject, and the selected route of administration.
  • the appropriate dosage can be readily determined by a person skilled in the art.
  • compositions may contain from 0.1% by weight, preferably from 5-60%, more preferably from 10-30% by weight, of a compound of invention, depending on the method of administration.
  • the optimal quantity and spacing of individual dosages of a compound of the invention will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the age and condition of the particular subject being treated, and that a physician will ultimately determine appropriate dosages to be used. This dosage may be repeated as often as appropriate. If side effects develop the amount and/or frequency of the dosage can be altered or reduced, in accordance with normal clinical practice.
  • the present invention provides methods for the production of 11-O-desmethylmacbecin analogues.
  • Macbecin can be considered to be biosynthesised in two stages.
  • the core-PKS genes assemble the macrolide core by the repeated assembly of 2-carbon units which are then cyclised to form the first enzyme-free intermediate “pre-macbecin”, see FIG. 1 .
  • a series of “post-PKS” tailoring enzymes e.g. P450 monooxygenases, methyltransferases, FAD-dependent oxygenases and a carbamoyltransferase
  • the 11-O-desmethylmacbecin analogues may be biosynthesised in a similar manner.
  • the present invention provides a method of producing 11-O-desmethylmacbecin analogues said method comprising:
  • post-PKS genes b) deleting or inactivating one or more post-PKS genes, wherein at least one of the post-PKS genes is mbcMT1, or a homologue thereof
  • step (a) by “macbecin or an analogue thereof” is meant macbecin or those analogues of macbecin that are embraced by the definitions of R 1 -R 4 .
  • step (b) deleting or inactivating one or more post-PKS genes, wherein at least one of the post-PKS genes is mbcMT1, or a homologue thereof will suitably be done selectively.
  • step b) comprises inactivating mbcMT1 (or a homologue thereof) by integration of DNA into the mbcMT1 gene (or a homologue thereof) such that functional MbcMT1 protein is not produced.
  • step b) comprises making a targeted deletion of the mbcMT1 gene, or a homologue thereof.
  • mbcMT1, or a homologue thereof is inactivated by site-directed mutagenesis.
  • the host strain of step a) is subjected to mutagenesis and a modified strain is selected in which one or more of the post-PKS enzymes are not functional, wherein at least one of these is MbcMT1.
  • the present invention also encompasses mutations of the regulators controlling the expression of mbcMT1, or a homologue thereof, a person of skill in the art will appreciate that deletion or inactivation of a regulator may have the same outcome as deletion or inactivation of the gene.
  • the strain of step b) is complemented with one or more of the genes that have been deleted or inactivated, not including mbcMT1 or a homologue thereof.
  • a method of selectively deleting or inactivating a post PKS gene comprises:
  • the macbecin-producing strain in step (i) is Actinosynnema mirum (A. mirum). In a further specific embodiment the macbecin-producing strain in step (ii) is Actinosynnema pretiosum ( A. pretiosum ).
  • additional post-PKS genes may also be deleted or inactivated in addition to mbcMT1.
  • FIG. 2 shows the activity of the post-PKS genes in the macbecin biosynthetic cluster. A person of skill in the art would thus be able to identify which additional post-PKS genes would need to be deleted or inactivated in order to arrive at a strain that will produce the compound(s) of interest.
  • mbcMT2 may be predicted to be in an operon with mbcMT1.
  • 11-O-Desmethylmacbecin analogues may be screened by a number of methods, as described herein, and in the circumstance where a single compound shows a favourable profile a strain can be engineered to make this compound preferably. In the unusual circumstance when this is not possible, an intermediate can be generated which is then biotransformed to produce the desired compound.
  • the present invention provides novel macbecin analogues generated by the selected deletion or inactivation of one or more post-PKS genes from the macbecin PKS gene cluster.
  • the present invention relates to novel 11-O-desmethylmacbecin analogues produced by the selected deletion or inactivation of at least mbcMT1, or a homologue thereof, from the macbecin PKS gene cluster.
  • mbcMT1, or a homologue thereof alone is deleted or inactivated.
  • other post-PKS genes in addition to mbcMT1 are additionally deleted or inactivated.
  • additional genes selected from the group consisting of: mbcM, mbcN, mbcP, mbcMT2 and mbcP450 are deleted or inactivated in the host strain.
  • additionally 1 or more of the post-PKS genes selected from the group consisting of: mbcM, mbcN, mbcP, mbcMT2 and mbcP450 are deleted or inactivated.
  • additionally 2 or more of the post-PKS genes selected from the group consisting of mbcM, mbcN, mbcP, mbcMT2 and mbcP450 are deleted or inactivated.
  • the post-PKS genes selected from the group consisting of mbcM, mbcN, mbcP, mbcMT2 and mbcP450 are deleted or inactivated.
  • additionally 4 or more of the post-PKS genes selected from the group consisting of mbcM, mbcN, mbcP, mbcMT2 and mbcP450 are deleted or inactivated.
  • mbcP, mbcP450, mbcMT1 and mbcMT2 are deleted.
  • a gene does not need to be completely deleted for it to be rendered non-functional, consequentially the term “deleted or inactivated” as used herein encompasses any method by which a gene is rendered non-functional including but not limited to: deletion of the gene in its entirety, deletion of part of the gene, inactivation by insertion into the target gene, site-directed mutagenesis which results in the gene either not being expressed or being expressed in an inactive form, mutagenesis of the host strain which results in the gene either not being expressed or being expressed in an inactive form (e.g. by radiation or exposure to mutagenic chemicals, protoplast fusion or transposon mutagenesis).
  • an active gene can be impaired chemically with inhibitors, for example metapyrone (alternative name 2-methyl-1,2-di(3-pyridyl-1-propanone), EP 0 627 009) and ancymidol are inhibitors of oxygenases and these compounds can be added to the production medium to generate analogues.
  • sinefungin is a methyl transferase inhibitor that can be used similarly but for the inhibition of methyl transferase activity in vivo (McCammon and Parks, 1981).
  • a method for the production of a 11-O-desmethylmacbecin analogue comprising:
  • an engineered strain in which one or more post-PKS genes including mbcMT1 have been deleted or inactivated is complemented by one or more of the post PKS genes from a heterologous PKS cluster including, but not limited to the clusters directing the biosynthesis of rifamycin, ansamitocin, geldanamycin or herbimycin.
  • all of the post-PKS genes may be deleted or inactivated and then one or more of the genes, but not including mbcMT1, or a homologue thereof, may then be reintroduced by complementation (e.g. at an attachment site, on a self-replicating plasmid or by insertion into a homologous region of the chromosome). Therefore, in a particular embodiment the present invention relates to methods for the generation of 11-O-desmethylmacbecin analogues, said method comprising:
  • one or more of the deleted post-PKS genes are reintroduced, provided that mbcMT1 is not one of the genes reintroduced.
  • 1 or more of the post-PKS genes selected from the group consisting of mbcM, mbcN, mbcP, mbcMT2 and mbcP450 are reintroduced.
  • 2 or more of the post-PKS genes selected from the group consisting of mbcM, mbcN, mbcP, mbcMT2 and mbcP450 are reintroduced.
  • 3 or more of the post-PKS genes selected from the group consisting of mbcM, mbcN, mbcP, mbcMT2 and mbcP450 are reintroduced.
  • 4 or more of the post-PKS genes selected from the group consisting of mbcM, mbcN, mbcP, mbcMT2 and mbcP450 are reintroduced.
  • mbcM, mbcN, mbcP, mbcMT2 and mbcP450 are reintroduced.
  • the strain in which mbcP, mbcP450, mbcMT1 and mbcMT2 are deleted is complemented by mbcMT2.
  • the strain in which mbcMT1 and mbcMT2 are deleted is complemented by mbcMT2.
  • the present invention includes the transfer of the macbecin biosynthetic gene cluster without mbcMT1 or with a non-functional mutant of mbcMT1, with or without resistance and regulatory genes, either otherwise complete or containing additional deletions, into a heterologous host.
  • the complete macbecin biosynthetic cluster can be transferred into a heterologous host, with or without resistance and regulatory genes, and it can then be manipulated by the methods described herein to delete or inactivate mbcMT1.
  • a preferred host cell strain is a prokaryote, more preferably an actinomycete or Escherichia coli , still more preferably include, but are not limited to Actinosynnema mirum ( A. mirum ), Actinosynnema pretiosum subsp. pretiosum ( A. pretiosum ), S. hygroscopicus, S. hygroscopicus sp., S. hygroscopicus var.
  • Streptomyces tsukubaensis Streptomyces coelicolor
  • Streptomyces lividans Saccharopolyspora erythraea, Streptomyces fradiae, Streptomyces avermitilis, Streptomyces cinnamonensis, Streptomyces rimosus, Streptomyces albus, Streptomyces griseofuscus, Streptomyces longisporoflavus, Streptomyces venezuelae, Streptomyces albus, Micromonospora sp., Micromonospora griseorubida, Amycolatopsis mediterranei or Actinoplanes sp. N902-109. Further examples include Streptomyces hygroscopicus subsp. geldanus and Streptomyces violaceusniger.
  • the entire biosynthetic cluster without mbcMT1 is transferred.
  • the entire PKS is transferred without any of the associated post-PKS genes, including mbcMT1.
  • some of the post-PKS genes, not including mbcMT1 can be introduced appropriately.
  • genes from other clusters such as the geldanamycin or herbimycin pathways can be introduced appropriately.
  • the entire macbecin biosynthetic cluster is transferred and then manipulated according to the description herein.
  • the 11-O-desmethylmacbecin analogue of the present invention may be further processed by biotransformation with an appropriate strain.
  • the appropriate strain either being an available wild type strain for example, but without limitation Actinosynnema mirum, Actinosynnema pretiosum subsp. pretiosum, S. hygroscopicus, S. hygroscopicus sp.
  • an appropriate strain may be a engineered to allow biotransformation with particular post-PKS enzymes for example, but without limitation, those encoded by mbcM, mbcN, mbcP, mbcMT2, mbcP450 (as defined herein), gdmN, gdmM, gdmL, gdmP, (Rascher et al., 2003) the geldanamycin O-methyl transferase, hbmN, hbmL, hbmP, (Rascher et al., 2005) herbimycin O-methyl transferases and further herbimycin mono-oxygenases, asm7, asm10, asm11, asm12, asm19 and asm21 (Cassady et al., 2004, Spiteller et al., 2003).
  • post-PKS enzymes for example, but without limitation, those encoded by mbcM, mbcN, mb
  • sequences are not in the public domain it is routine to those skilled in the art to acquire such sequences by standard methods.
  • sequence of the gene encoding the geldanamycin O-methyl transferase is not in the public domain, but one skilled in the art could generate a probe, either a heterologous probe using a similar O-methyl transferase, or a homologous probe by designing degenerate primers from available homologous genes to carry out Southern blots on a geldanamycin producing strain and thus acquire this gene to generate biotransformation systems.
  • the strain may have had one or more of its native polyketide clusters deleted, either entirely or in part, or otherwise inactivated, so as to prevent the production of the polyketide produced by said native polyketide cluster.
  • Said engineered strain may be selected from the group including, for example but without limitation, Actinosynnema mirum, Actinosynnema pretiosum subsp. pretiosum, S. hygroscopicus, S. hygroscopicus sp., S. hygroscopicus var.
  • the present invention provides host strains which naturally produce macbecin or analogue thereof, in which the mbcMT1 gene, or a homologue thereof, has been deleted or inactivated such that it thereby produces 11-O-desmethylmacbecin or an analogue thereof (e.g. a 11-O-desmethylmacbecin analogue as defined by formula (I)) and their use in the production of 11-O-desmethylmacbecin or analogues thereof.
  • 11-O-desmethylmacbecin or analogue thereof e.g. a 11-O-desmethylmacbecin analogue as defined by formula (I)
  • the present invention provides a genetically engineered strain which naturally produces macbecin in its unaltered state, said strain having one or more post-PKS genes from the macbecin PKS gene cluster deleted wherein one of said deleted or inactivated post-PKS genes is mbcMT1, or a homologue thereof.
  • the invention embraces all products of the inventive processes described herein.
  • the gene cluster was sequenced from Actinosynnema pretiosum subsp. pretiosum however, a person of skill in the art will appreciate that there are alternative strains which produce macbecin, for example but without limitation Actinosynnema mirum .
  • the macbecin biosynthetic gene cluster from these strains may be sequenced as described herein for Actinosynnema pretiosum subsp. pretiosum , and the information used to generate equivalent strains.
  • Compounds of the invention are advantageous in that they may be expected to have one or more of the following properties: good activity against one or more different cancer sub-types compared with the parent compound; good toxicological profile such as good hepatotoxicity profile, good nephrotoxicity, good cardiac safety; good water solubility; good metabolic stability; good formulation ability; good bioavailability; good pharmacokinetic or pharmacodynamic properties such as tight binding to Hsp90, fast on-rate of binding to Hsp90 and/or good brain pharmacokinetics; good cell uptake; and low binding to erythrocytes.
  • Actinosynnema pretiosum subsp. pretiosum ATCC 31280 U.S. Pat. No. 4,315,989
  • Actinosynnema mirum DSM 43827 KCC A-0225, Watanabe et al., 1982
  • Methods used herein were adapted from these and are as follows for culturing of broths in tubes or flasks in shaking incubators, variations to the published protocols are indicated in the examples.
  • Strains were grown on ISP2 agar (Medium 3, Shirling, E. B.
  • 4,187,292 was inoculated with 2.5%-10% of the seed culture and incubated with shaking between 200 and 300 rpm with a 5 or 2.5 cm throw initially at 28° C. for 24 h followed by 26° C. for four to six days. The culture was then harvested for extraction.
  • LCMS was performed using an integrated Agilent HP1100 HPLC system in combination with a Bruker Daltonics Esquire 3000+ electrospray mass spectrometer operating in positive and/or negative ion mode. Chromatography was achieved over a Phenomenex Hyperclone column (C 18 BDS, 3u, 150 ⁇ 4.6 mm) eluting over 11 min at a flow rate of 1 mL/min with a linear gradient from acetonitrile+0.1% formic acid/water+0.1% formic acid (40/60) to acetonitrile+0.1% formic acid/water+0.1% formic acid (80/20). UV spectra were recorded between 190 and 400 nm, with extracted chromatograms taken at 210, 254 and 276 nm. Mass spectra were recorded between 100 and 1500 amu.
  • NMR spectra were recorded on a Bruker Advance 500 spectrometer at 298 K operating at 500 MHz and 125 MHz for 1 H and 13 C respectively. Standard Bruker pulse sequences were used to acquire 1 H- 1 H COSY, APT, HMBC and HMQC spectra. NMR spectra were referenced to the residual proton or standard carbon resonances of the solvents in which they were run.
  • Water solubility may be tested as follows: A 10 mM stock solution of the 11-O-desmethylmacbecin analogue is prepared in 100% DMSO at room temperature. Triplicate 0.01 mL aliquots are made up to 0.5 mL with either 0.1 M PBS, pH 7.3 solution or 100% DMSO in amber vials. The resulting 0.2 mM solutions are shaken in the dark, at room temperature on an IKA® vibrax VXR shaker for 6 h, followed by transfer of the resulting solutions or suspensions into 2 mL Eppendorf tubes and centrifugation for 30 min at 13200 rpm. Aliquots of the supernatant fluid are then analysed by LCMS as described above.
  • Oncotest cell lines are established from human tumor xenografts as described by Roth et al., (1999). The origin of the donor xenografts was described by Fiebig et al., (1999). Other cell lines are either obtained from the NCI (DU145, MCF-7) or purchased from DSMZ, Braunschweig, Germany.
  • a modified propidium iodide assay was used to assess the effects of the test compound(s) on the growth of human tumour cell lines (Dengler et al., (1995)).
  • cells were harvested from exponential phase cultures by trypsinization, counted and plated in 96 well flat-bottomed microtitre plates at a cell density dependent on the cell line (5-10.000 viable cells/well). After 24 h recovery to allow the cells to resume exponential growth, 0.010 mL of culture medium (6 control wells per plate) or culture medium containing the 11-O-desmethylmacbecin analogue were added to the wells. Each concentration was plated in triplicate. Compounds were applied in two concentrations (1 ⁇ g/mL and 10 ⁇ g/mL). Following 4 days of continuous exposure, cell culture medium with or without test compound was replaced by 0.2 mL of an aqueous propidium iodide (PI) solution (7 mg/L).
  • PI propidium iodide
  • Genomic DNA was isolated from Actinosynnema pretiosum (ATCC 31280) and Actinosynnema mirum (DSM 43827, ATCC 29888) using standard protocols described in Kieser et al., (2000). DNA sequencing was carried out by the sequencing facility of the Biochemistry Department, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW using standard procedures.
  • the DIG-labelled gdmN DNA fragment was used as a heterologous probe. Using the gdmN generated probe and genomic DNA isolated from A. pretiosum 2112 an approximately 8 kb EcoRI fragment was identified in Southern blot analysis. The fragment was cloned into Litmus 28 applying standard procedures and transformants were identified by colony hybridization. The clone p3 was isolated and the approximately 7.7 kb insert was sequenced. DNA isolated from clone p3 was digested with EcoRI and EcoRI/SacI and the bands at around 7.7 kb and at about 1.2 kb were isolated, respectively. Labelling reactions were carried out according to the manufacturers' protocols.
  • Cosmid libraries of the two strains named above were created using the vector SuperCos 1 and the Gigapack III XL packaging kit (Stratagene) according to the manufacturers' instructions. These two libraries were screened using standard protocols and as a probe, the DIG-labelled fragments of the 7.7 kb EcoRI fragment derived from clone p3 were used. Cosmid 52 was identified from the cosmid library of A. pretiosum and submitted for sequencing to the sequencing facility of the Biochemistry Department of the University of Cambridge. Similarly, cosmid 43 and cosmid 46 were identified from the cosmid library of A. mirum . All three cosmids contain the 7.7 kb EcoRI fragment as shown by Southern Blot analysis.
  • sequence information of cosmid 52 was also used to create probes derived from DNA fragments amplified by primers BIOSG130 5′-CCAACCCCGCCGCGTCCCCGGCCGCGCGCCGAACACG-3′ (SEQ ID NO: 5) and BIOSG131 5′-GTCGTCGGCTACGGGCCGGTGGGGCAGCTGCTGT-5′ (SEQ ID NO: 6) as well as BIOSG132 5′-GTCGGTGGACTGCCCTGCGCCTGATCGCCCTGCGC-3′ (SEQ ID NO: 7) and BIOSG133 5′-GGCCGGTGGTGCTGCCCGAGGACGGGGAGCTGCGG-3′ (SEQ ID NO: 8) which were used for screening the cosmid library of A. pretiosum.
  • Cosmids 311 and 352 were isolated and cosmid 352 was sent for sequencing.
  • Cosmid 352 contains an overlap of approximately 2.7 kb with cosmid 52.
  • To screen for further cosmids an approximately 0.6 kb PCR fragment was amplified using primers BIOSG136 5′-CACCGCTCGCGGGGGTGGCGCGGCGCACGACGTGG CTGC-3′ (SEQ ID NO: 9) and BIOSG 137 5′-CCTCCTCGGACAGCGCGATCAGCGCCGCGC ACAGCGAG-3′ (SEQ ID NO: 10) and cosmid 311 as template applying standard protocols.
  • the cosmid library of A. pretiosum was screened and cosmid 410 was isolated.
  • Oligos gpOMTa SEQ ID NO: 12
  • gpOMTb SEQ ID NO: 13
  • a 5′ extension was designed in each oligo to introduce an XbaI site (underlined) to aid cloning of the amplified fragment ( FIG. 3 ).
  • the amplified PCR product (PCRgp24, SEQ ID NO: 14, FIG.
  • gpOMTa (SEQ ID NO: 12) 5′- TCTAGA ACGAGCACACCTACGAGCAGTTCGAGAAGT -3′ gpOMTb (SEQ ID NO: 13) 5′- TCTAGA GATCTCCAGGGTCTCCCGCCAAGTGCGTTC -3′ 2.2 Transformation of Actinosynnema pretiosum subsp. pretiosum
  • Escherichia coli ET12567 harbouring the plasmid pUZ8002, was transformed with pGP24 by electroporation to generate the E. coli donor strain for conjugation.
  • This strain was used to transform Actinosynnema pretiosum subsp. pretiosum by vegetative conjugation (Matsushima et al., 1994).
  • Exconjugants were plated on Medium 4 and incubated at 28° C. Plates were overlayed after 24 h with 50 mg/L apramycin and 25 mg/L nalidixic acid. As pGP24 is unable to replicate in Actinosynnema pretiosum subsp.
  • any apramycin resistant colonies were anticipated to be transformants that contained plasmid integrated into the mbcMT1 gene of the chromosome by homologous recombination via the plasmid borne mbcMT1 internal fragment. This resulted in two truncated copies of the mbcMT1 gene on the chromosome.
  • Four exconjugants were isolated and tested for the production of macbecin analogues.
  • Colonies were patched onto Medium 4 (with 50 mg/L apramycin and 25 mg/L nalidixic acid).
  • a 6 mm circular plug from each patch was used to inoculate individual 50 mL falcon tubes containing 10 mL seed medium (variant of Medium 1-2% glucose, 3% soluble starch, 0.5% corn steep solids, 1% soybean flour, 0.5% peptone, 0.3% sodium chloride, 0.5% calcium carbonate) plus 50 mg/L apramycin.
  • seed medium variant of Medium 1-2% glucose, 3% soluble starch, 0.5% corn steep solids, 1% soybean flour, 0.5% peptone, 0.3% sodium chloride, 0.5% calcium carbonate
  • These seed cultures were incubated for 2 days at 28° C., 200 rpm with a 5 cm throw. These were then used to inoculate (5% v/v) fermentation medium (Medium 2) and were grown at 28° C. for 24 hours and then at 26° C. for a further 5 days. Metabolites were extracted from these according
  • BIOT-3820 One isolate produced no macbecin and two novel compounds with characteristic macbecin like quinone chromophores and was designated BIOT-3820.
  • the major component displayed characteristic ions with m/z 529.4 [M ⁇ H] ⁇ and 553.4 [M+Na] + , consistent with the structure 11-O-desmethyl-15-O-desmethylmacbecin 14 (molecular formulae C 28 H 38 N 2 O 8 ).
  • the second, later eluting component displayed ions with m/z of 513.4 [M ⁇ H] ⁇ , consistent with the structure 11-O-desmethyl-15-desmethoxymacbecin 15 (molecular formulae C 28 H 38 N 2 O 7 ).
  • BIOT-3820 an Actinosynnema pretiosum Strain in which the O-methyltransferase mbcMT1 is Disrupted
  • Vegetative cultures were prepared by removing two agar plugs, 6 mm in diameter, from a MAM plate (Medium 4) and inoculating them into 4 ⁇ 30 mL medium 1 in 250 mL shake flasks containing 50 mg/L apramycin. The flasks were incubated at 28° C., 200 rpm (5 cm throw) for 48 h.
  • Vegetative cultures were inoculated at 5% v/v into 8 ⁇ 200 mL production medium (medium 2) in 2 L shake flasks. Cultivation was carried out for 1 day at 28° C. followed by 5 days at 26° C., 200 rpm (5 cm throw).
  • the fermentation broth (1 L, pink colour) was extracted three times with an equal volume of ethyl acetate.
  • the organic extracts were combined and the solvent removed in vacuo to yield 2.5 g of an oily residue.
  • This was dissolved in methanol (15 mL) and a solution of FeCl 3 (1%, 200 mL) added.
  • This was extracted with ethyl acetate (3 ⁇ 200 mL), the organic extracts combined and the solvent removed in vacuo to yield an oily residue (1.7 g).
  • the residue was then chromatographed over Silica gel 60 eluting with a step gradient from CHCl 3 to CHCl 3 :methanol (97:3) and collecting fractions of ca. 200 mL.
  • Oligos KOmbc450F (SEQ ID NO: 15) and KOmbc450R (SEQ ID NO: 16) were used in a standard PCR reaction using cosmid52 (from example 1) as a template to amplify a 518 bp internal DNA fragment of mbcP450 (SEQ ID NO: 17) FIG. 5 .
  • the resulting DNA fragment was ligated into SmaI digested pUC19 giving plasmid pNG9-20/06/05. Insertion was confirmed via restriction analysis and sequencing. Plasmid pNG9-20/06/05 was then digested using enzymes EcoRI and HindIII. The resulting 571 bp DNA fragment was ligated into EcoRI/HindIII digested plasmid pKC1132 ( FIG. 6 ). Plasmid pNGmbcP450 was isolated and confirmed via restriction enzyme analysis.
  • KOmbc450F (SEQ ID NO: 15): 5′- TTCGTGCAGCGGATCGTCGA -3′
  • KOmbc450R (SEQ ID NO: 16): 5′- ATCCCGGTGTGCGAGATCGT -3′ 3.2. Transformation of Actinosynnema pretiosum subsp. pretiosum
  • Escherichia coli ET12567 harbouring plasmid pUZ8002 was used to transform pNGmbcP450 by electroporation to generate the E. coli donor strain for conjugation.
  • This strain was used for conjugation experiments in combination with Actinosynnema pretiosum subsp. pretiosum (Matsushima et al, 1994).
  • Conjugated cells were plated on Medium 4 (MAM medium) and incubated at 28° C. Plates were overlaid after 16 h with 50 mg/L apramycin and 25 mg/L nalidixic acid.
  • Colonies were patched onto Medium 4 containing 50 mg/L apramycin and 25 mg/L nalidixic acid, and then re-patched similarly. Each colony was used to inoculate 10 mL of Medium 1 (seed medium) plus 50 mg/L apramycin. Cultures were incubated for 2 days at 28° C., 200 rpm and cells harvested. Genomic DNA was isolated from each sample and plasmid integration was confirmed by Southern Blot analysis using standard techniques.
  • a 6 mm circular plug from each patch was used to inoculate individual 50 mL falcon tubes containing 10 mL of Medium 1 (seed medium) plus 50 mg/L apramycin. These seed cultures were incubated for 2 days at 28° C., 200 rpm. 0.5 mL of these cultures was used to inoculate 10 mL of Medium 2 (production medium). Cultures were grown at 28° C. for 24 hours and then at 26° C. for a further 5 days. Secondary metabolites were extracted from these cultures and samples were assessed for production of macbecin analogues by HPLC using Method 1 as described above. One isolate produced no macbecin and two novel compounds with characteristic macbecin like quinone chromophores and was designated BIOT-3825.
  • Oligos BioSG138 SEQ ID NO: 18
  • BioSG139 SEQ ID NO: 19
  • Oligos BioSG138 SEQ ID NO: 18
  • BioSG139 SEQ ID NO: 19
  • the amplified PCR product, PCR1 SEQ ID NO: 20, FIG. 7
  • PCR1 was cloned into vector Litmus28 previously linearised with EcoRV using standard techniques. Plasmid Lit28PCR1 no6 was isolated and confirmed by DNA sequence analysis. The analysis was completed using the sequencing primer BioSG150 (SEQ ID NO: 21).
  • BioSG138 (SEQ ID NO: 18) 5′-GG AAGCTT TCGGTAATGGGGAGACTCGACGCCGCCTGAC-3′ BioSG139 (SEQ ID NO: 19) 5′-G GGATCC CCGAACACCCGTAACCACGCGGTGGCGTCCCCC-3′ BioSG150 (SEQ ID NO: 21) 5′-CAGCAGGAGTTCCCGCAAGAGTTGGAGCGC-3′ 4.2 Cloning of DNA Homologous to the Upstream Flanking Region of mbcMT1.
  • Oligos BioSG140 SEQ ID NO: 22
  • BioSG141 SEQ ID NO: 23
  • PCR2 SEQ ID NO: 24; FIG. 8
  • Plasmid Lit28PCR2 no8 was isolated and confirmed by DNA sequence analysis. The analysis was completed using the sequencing primer BioSG152 (SEQ ID NO: 25).
  • BioSG140 (SEQ ID NO: 22) 5′-G GGATCC GGGAACGGCCTTTCGGGGTCGGCTTGCGGGAGG-3′ BioSG141 (SEQ ID NO: 23) 5′-GG GAATTC CCCCGGAGAAAGGCCGCCGCAGTGTTCAC-3′ BioSG152 (SEQ ID NO: 25) 5′-CCTCGTGGTCGGAGTAGGGCAGGCCCAGGACGG-3′ 4.3 Isolation of pKC1132PCR1
  • Plasmid Lit28PCR1 no6 was digested with HindIII/BamHI, the approximately 2.5 kb DNA insert was isolated and cloned into pKC1132 (Bierman et al., 1992) previously treated with HindIII/BamHI using standard techniques. Plasmid pKC1132PCR1 was isolated and confirmed by restriction digests.
  • Plasmid Lit28PCR2 no8 was digested with BamHI/EcoRI, the about 2.5 kb DNA insert was isolated and cloned into pKC1132PCR1 previously treated with BamHI/EcoRI using standard techniques. Plasmid pKC1132PCR1PCR2 ( FIG. 9 ) was isolated and confirmed by restriction digests and sequence analysis.
  • pretiosum 4.5 Transformation of Actinosynnema pretiosum subsp. pretiosum
  • Escherichia coli ET12567 harbouring the plasmid pUZ8002 was used to transform pKC1132PCR1PCR2 by electroporation to generate the E. coli donor strain for conjugation.
  • This strain was used for conjugation experiments in combination with Actinosynnema pretiosum subsp. pretiosum (Matsushima et al, 1994).
  • Conjugated cells were plated on medium 4 (MAM medium) and incubated at 28° C. Plates were overlayed after 24 h with 50 mg/L apramycin and 25 mg/L nalidixic acid. Genomic DNA was isolated from apramycin resistant clones and plasmid integration was confirmed by Southern Blot analysis using standard techniques.
  • Apramycin sensitive clones were re-patched to confirm the loss of the antibiotic marker.
  • Deletion mutants were patched onto MAM medium and grown at 28° C. for four days.
  • a 6 mm circular plug from each patch was used to inoculate individual 50 mL falcon tubes containing 10 mL of Medium 1 (seed medium). These seed cultures were incubated for 2 days at 28° C., 200 rpm with a 2 inch throw. These were then used to inoculate (0.5 mL into 10 mL) Medium 2 (production medium) and were grown at 28° C. for 24 hours and then at 26° C. for a further 5 days. Secondary metabolites were extracted from these cultures and samples were assessed for production of macbecin analogues by HPLC as described in General Methods. One isolate was designate BIOT-3848.
  • Oligos BioSG142 SEQ ID NO: 26
  • BioSG147 SEQ ID NO: 27
  • PCR mbcMT2 SEQ ID NO: 28; FIG. 10
  • cosmid 52 from example 1
  • the amplified PCR product was cloned into vector Litmus28 previously linearised with EcoRV using standard techniques. Plasmid Lit28 mbcMT2 no17 was isolated and confirmed by DNA sequence analysis.
  • Plasmid Lit28 mbcMT2 no17 was digested with NdeI/XbaI and the about 0.8 kb insert DNA fragment was isolated and cloned into NdeI/XbaI treated vector pGP9. Plasmid pGP9 mbcMT2 was isolated using standard techniques. The construct was confirmed by restriction digest analysis.
  • BioSG142 (SEQ ID NO: 26) 5′-GG TCTAGA GGTCACGGGTGTTCGGCTACTGCTAGGAAGCAGCC-3′
  • BioSG147 (SEQ ID NO: 27) 5′-GG CATATG AGGCCCGTCCCCGAGGCCGTGGGCCGCC-3′ 4.9 Complementation of A. pretiosum ⁇ MT1 MT2 no13 with pGP9 mbcMT2
  • pretiosum ⁇ MT1 MT2 no13 using plasmid pGP9 mbcMT2 were carried out as described above (4.5) and apramycin resistant colonies were isolated.
  • the production of macbecin related compounds was assessed as described in General Methods.
  • the plates were used to inoculate 250 mL conical flasks containing 30 mL of Medium 1 (seed medium) plus 50 mg/L apramycin with two 6 mm circular plugs each. These seed cultures were incubated for 2 days at 28° C. as described in General Methods.
  • the cultures were then used to inoculate (15 mL into 300 mL) 8 ⁇ 2 litre conical flasks each containing 300 mL of Medium 2 (production medium) and were grown at 26° C. for 6 days as described in General Methods.
  • the cultures were pooled and secondary metabolites were extracted from these cultures as described in General Methods.
  • Oligos Is4deI1 SEQ ID NO: 29
  • Is4deI2a SEQ ID NO: 30
  • a 5′ extension was designed in oligo Is4deI2a to introduce an AvrII site to aid cloning of the amplified fragment ( FIG. 11 ).
  • the amplified PCR product (1+2a, SEQ ID NO: 31, FIG.
  • Is4deI1 (SEQ ID NO: 29) 5′- GGTCACTGGCCGAAGCGCACGGTGTCATGG -3′
  • Is4deI2a (SEQ ID NO: 30) 5′- CCTAGGCGACTACCCCGCACTACTACACCGAGCAGG -3′ 5.2 Cloning of DNA Homologous to the Upstream Flanking Region of mbcM
  • Oligos Is4deI3b SEQ ID NO: 32
  • Is4deI4 SEQ ID NO: 33
  • a 5′ extension was designed in oligo Is4deI3b to introduce an AvrII site to aid cloning of the amplified fragment ( FIG. 11 ).
  • the amplified PCR product (3b+4, SEQ ID NO: 34, FIG.
  • Is4deI3b (SEQ ID NO: 32) 5′- CCTAGGAACGGGTAGGCGGGCAGGTCGGTG -3′
  • Is4deI4 (SEQ ID NO: 33) 5′- GTGTGCGGGCCAGCTCGCCCAGCACGCCCAC -3′
  • the products 1+2a and 3b+4 were cloned into pUC19 to utilise the HindIII and BamHI sites in the pUC19 polylinker for the next cloning step.
  • the 1621 bp AvrII/HindIII fragment from pLSS1+2a and the 1543 bp AvrII/BamHI fragment from pLSS3b+4 were cloned into the 3556 bp HindIII/BamHI fragment of pKC1132 to make pLSS315.
  • pLSS315 therefore contained a HindIII/BamHI fragment encoding DNA homologous to the flanking regions of the desired four ORF deletion region fused at an AvrII site ( FIG. 11 ).
  • pretiosum 5.3 Transformation of Actinosynnema pretiosum subsp. pretiosum
  • Escherichia coli ET12567 harbouring the plasmid pUZ8002 was transformed with pLSS315 by electroporation to generate the E. coli donor strain for conjugation.
  • This strain was used to transform Actinosynnema pretiosum subsp. pretiosum by vegetative conjugation (Matsushima et al, 1994)
  • Exconjugants were plated on MAM medium (1% wheat starch, 0.25% corn steep solids, 0.3% yeast extract, 0.3% calcium carbonate, 0.03% iron sulphate, 2% agar) and incubated at 28° C. Plates were overlayed after 24 h with 50 mg/L apramycin and 25 mg/L nalidixic acid.
  • a single clone of this third type was assigned the identifier Actinosynnema pretiosum :pLSS315#5; BIOT-3827.
  • Vegetative cultures were prepared by removing two agar plugs, 6 mm in diameter, from a MAM plate (Medium 4) and inoculating them into 8 ⁇ 30 mL Medium 1 in 250 mL shake flasks containing 50 mg/L apramycin. The flasks were incubated at 28° C., 200 rpm (5 cm throw) for 48 h.
  • Vegetative cultures were inoculated at 5% v/v into 12 ⁇ 225 mL production medium (Medium 2) in 2 L shake flasks. Cultivation was carried out for 1 day at 28° C. followed by 5 days at 26° C., 200 rpm (5 cm throw).
  • the fermentation broth ( ⁇ 2 L, pink colour) was extracted three times with an equal volume of ethyl acetate.
  • the organic extracts were combined and the solvent removed in vacuo to yield 2.7 g of an oily residue.
  • This was dissolved in methanol (15 mL) and a solution of FeCl 3 (1%, 200 mL) added.
  • This was extracted with ethyl acetate (3 ⁇ 200 mL), the organic extracts combined and the solvent removed in vacuo to yield an oily residue (1.75 g).
  • the residue was then chromatographed over Silica gel 60 eluting with a step gradient from CHCl 3 :MeOH (99:1) to CHCl 3 :methanol (95:5) and collecting fractions of ca.
  • Genomic DNA was isolated from the six exconjugants and digested and analysed by Southern Blot. The blot showed that in five out of the six isolates integration had occurred in the RHS region of homology and in one of the six isolates homologous integration had occurred in the left hand side region.
  • the desired mutant strains have a deletion of 3892 bp of the macbecin cluster containing the genes mbcP, mbcP450, mbcMT1 and mbcMT2.
  • Deletion mutants were patched onto MAM medium and grown at 28° C. for four days. A 6 mm circular plug from each patch was used to inoculate individual 50 mL falcon tubes containing 10 mL seed medium (2% glucose, 3% soluble starch, 0.5% corn steep solids, 1% soybean flour, 0.5% peptone, 0.3% sodium chloride, 0.5% calcium carbonate). These seed cultures were incubated for 2 days at 28° C., 200 rpm with a 2 inch throw.
  • seed medium 2% glucose, 3% soluble starch, 0.5% corn steep solids, 1% soybean flour, 0.5% peptone, 0.3% sodium chloride, 0.5% calcium carbonate.
  • BIOT-3863 an Actinosynnema pretiosum strain in which the mbcP has been Interrupted by Insertion of a Plasmid
  • Oligos KOmbcPF (SEQ ID NO: 35) and KOmbcPR (SEQ ID NO: 36) were used in a standard PCR reaction using cosmid52 (from example 1) as a template to amplify a 526 bp internal DNA-fragment of mbcP (SEQ ID NO: 37; FIG. 14 ) from Actinosynnema pretiosum .
  • the resulting DNA fragment was ligated into the SmaI digested pUC19 giving plasmid pNG4-06/07/05. Insertion was confirmed via restriction analysis and sequencing.
  • Plasmid pNG4-06/07/05 was then digested using enzymes EcoRI and HindIII and the resulting 579 bp DNA fragment was ligated into the previously EcoRI/HindIII digested plasmid pKC1132 ( FIG. 15 ). Plasmid pNGmbcP was isolated and confirmed via restriction analysis.
  • KOmbcPF (SEQ ID NO: 35): 5′-GCATGGTCGCCGGGCACTTC-3′
  • KOmbcPR (SEQ ID NO: 36): 5′-TCGACGGCGTTGGCGAAACC -3′ 6.2. Transformation of Actinosynnema pretiosum subsp. pretiosum
  • Escherichia coli ET12567 harbouring plasmid pUZ8002, was transformed with pNGmbcP by electroporation to generate the E. coli donor strain for conjugation.
  • This strain was used for conjugation experiments in combination with Actinosynnema pretiosum subsp. pretiosum (Matsushima et al, 1994).
  • Conjugated cells were plated on medium 4 (MAM medium) and incubated at 28° C. Plates were overlaid after 16 h with 50 mg/L apramycin and 25 mg/L nalidixic acid. Colonies were patched onto medium 4 containing 50 mg/L apramycin and 25 mg/L nalidixic acid, and then re-patched similarly.
  • a 6 mm circular plug from each patch was used to inoculate individual 50 mL falcon tubes containing 10 mL of medium 1 (seed medium) plus 50 mg/L apramycin. These seed cultures were incubated for 2 days at 28° C., 200 rpm. 0.5 mL of these cultures was then used to inoculate 10 mL of medium 2 (production medium). Cultures were grown at 28° C. for 24 hours and then at 26° C. for a further 5 days. Secondary metabolites were extracted from these cultures and samples were assessed for production of macbecin analogues by HPLC as described in General Methods.
  • test compounds for anticancer activity in a panel of human tumour cell lines in a monolayer proliferation assay was carried out as described in the general methods using a modified propidium iodide assay.

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CN107881137A (zh) * 2017-09-25 2018-04-06 辽宁斯韦尔生物科技有限公司 增强转录水平的高产安丝菌素菌株及其制备方法
US20210361292A1 (en) * 2018-07-02 2021-11-25 Tulavi Therapeutics, Inc. Methods for in situ formed nerve cap with rapid release

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WO2010138869A1 (fr) 2009-05-29 2010-12-02 The Trustees Of Columbia University In The City Of New York Modulation de la phospholipase d pour le traitement des maladies dégénératives du système nerveux

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US4421687A (en) * 1981-09-26 1983-12-20 Takeda Chemical Industries, Limited Macbecin derivatives
US20040077058A1 (en) * 2002-06-14 2004-04-22 Hutchinson Richard C. Recombinant polynucleotides encoding pro-geldanamycin producing polyketide synthase and accessory proteins, and uses thereof

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EP0664702A1 (fr) * 1992-10-14 1995-08-02 THE GOVERNMENT OF THE UNITED STATES OF AMERICA as represented by the SECRETARY OF THE DEPARTMENT OF HEALTH AND HUMAN SERVICES Activite tumoricide des ansamycines benzoquinonoides contre le cancer de la prostate et les malignites neurales primitives
CA2218523A1 (fr) * 1995-05-02 1996-11-07 William J. Welch Induction de la thermotolerance a l'aide d'ansamycines benzoquinonoides

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US4421687A (en) * 1981-09-26 1983-12-20 Takeda Chemical Industries, Limited Macbecin derivatives
US4512975A (en) * 1981-09-26 1985-04-23 Takeda Chemical Industries, Ltd. Macbecin derivatives and their production
US20040077058A1 (en) * 2002-06-14 2004-04-22 Hutchinson Richard C. Recombinant polynucleotides encoding pro-geldanamycin producing polyketide synthase and accessory proteins, and uses thereof

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CN107881137A (zh) * 2017-09-25 2018-04-06 辽宁斯韦尔生物科技有限公司 增强转录水平的高产安丝菌素菌株及其制备方法
US20210361292A1 (en) * 2018-07-02 2021-11-25 Tulavi Therapeutics, Inc. Methods for in situ formed nerve cap with rapid release

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